The tautomerase superfamily consists of structurally homologous enzymes based on a beta-alpha-beta motif whose members use Pro-i as the general base in tautomerization and isomerization reactions. The long-term goal of this research is to determine the molecular and structural basis for catalysis and specificity in the tautomerase superfamily. This research has implications for our understanding of fundamental enzymatic reactions and the evolution of enzymes. In addition, these studies will assist in determining the range of metabolic capabilities for various pathogenic organisms, potentially leading to the development of new drugs, and facilitate bio-remediation efforts. The focus of this application will be representative members of the 4-oxalocrotonate tautomerase (4-OT) family. These enzymes range in size from 61-79 amino acids per monomer and all have an amino-terminal proline. The principal investigator's group has recently discovered structural and mechanistic diversity in this family. This diversity suggests that Nature used these short sequences (encoding a simple beta-alpha-beta motif) to create new structures and activities. The major specific aims will be to determine the mechanisms and structures for 1) 3-chloroacrylic acid dehalogenase which may use Pro-1 to activate water for an addition reaction, 2) malonate semialdehyde decarboxylase, which may use Pro-1 in a Schiff base mechanism to facilitate decarboxylation, 3) a tautomerase, which has low level isomerase and dehalogenase activities, 4) a dimeric 4-OT homologue, and 5) two closely related homologues that have tautomerase and isomerase activities but lack the conserved active site residues (except Pro-1) found in the parent member, 4-OT. Finally, experiments are proposed to improve the low-level activities and to manipulate the oligomer state by rational design. These studies will provide a better understanding of the structure/function relationships for each enzyme. A comparison of the strategies and active site structures will provide signatures for each enzymatic activity, shed light on how these activities evolved, and assist in the assignment of function. As a result, the underlying principles used in this system will be identified so that Nature's processes could be mimicked to create new activities and structures using the beta-a-beta motif.